Kluyveromyces marxianus has been widely used in food industries and inulinase produced by the yeast has many applications. In order to greatly enhance inulinase production by the yeast, it is very important to both derepress glucose repression and overexpress the native inulinase gene. The MIG1 gene in the yeast K. marxianus KM-0 was disrupted. It was found that a glucose-derepressed mutant (KM-69) of K. marxianus KM-0 could produce 94.6 units of inulinase activity per ml in the inulin medium within 36 h. In order to further increase inulinase production, the native inulinase gene was over-expressed in the glucose-derepressed mutant. During the 10-I fermentation, the recombinant strain KM-526 yielded 133.5 units of inulinase activity per ml within very short time (24h). The results also showed that inulinase produced by the recombinant strain KM-526 could more effectively convert inulin into monosaccharides than that produced by K. marxianus KM-0. The results indicate that it is very important to delete the MIG1 gene in K. marxianus KM-0 and over-express the native INU1 gene in order to further enhance inulinase yield within the short time by this yeast and for efficient hydrolysis of inulin. The recombinant strain KM-526 and its inulinase have highly potential applications in food industries. (C) 2014 Elsevier Ltd. All rights reserved.